A Comprehensive Overview of the Genes and Functions Required for Lettuce Infection by the Hemibiotrophic Phytopathogen Xanthomonas hortorum pv. vitians

ABSTRACT The successful infection of a host plant by a phytopathogenic bacterium depends on a finely tuned molecular cross talk between the two partners. Thanks to transposon insertion sequencing techniques (Tn-seq), whole genomes can now be assessed to determine which genes are important for the fitness of several plant-associated bacteria in planta. Despite its agricultural relevance, the dynamic molecular interaction established between the foliar hemibiotrophic phytopathogen Xanthomonas hortorum pv. vitians and its host, lettuce (Lactuca sativa), remains completely unknown. To decipher the genes and functions mobilized by the pathogen throughout the infection process, we conducted a Tn-seq experiment in lettuce leaves to mimic the selective pressure occurring during natural infection. This genome-wide screening identified 170 genes whose disruption caused serious fitness defects in lettuce. A thorough examination of these genes using comparative genomics and gene set enrichment analyses highlighted that several functions and pathways were highly critical for the pathogen’s survival. Numerous genes involved in amino acid, nucleic acid, and exopolysaccharide biosynthesis were critical. The xps type II secretion system operon, a few TonB-dependent transporters involved in carbohydrate or siderophore scavenging, and multiple genes of the carbohydrate catabolism pathways were also critical, emphasizing the importance of nutrition systems in a nutrient-limited environment. Finally, several genes implied in camouflage from the plant immune system and resistance to immunity-induced oxidative stress were strongly involved in host colonization. As a whole, these results highlight some of the central metabolic pathways and cellular functions critical for Xanthomonas host adaptation and pathogenesis. IMPORTANCE Xanthomonas hortorum was recently the subject of renewed interest, as several studies highlighted that its members were responsible for diseases in a wide range of plant species, including crops of agricultural relevance (e.g., tomato and carrot). Among X. hortorum variants, X. hortorum pv. vitians is a reemerging foliar hemibiotrophic phytopathogen responsible for severe outbreaks of bacterial leaf spot of lettuce all around the world. Despite recent findings, sustainable and practical means of disease control remain to be developed. Understanding the host-pathogen interaction from a molecular perspective is crucial to support these efforts. The genes and functions mobilized by X. hortorum pv. vitians during its interaction with lettuce had never been investigated. Our study sheds light on these processes by screening the whole pathogen genome for genes critical for its fitness during the infection process, using transposon insertion sequencing and comparative genomics.

This manuscript has presented the results of an interesting transposon sequencing study to address those genes that are involved in colonization of lettuce leaves by a Xanthomonas pathogen. The study seems to have been done quite well, and the results clearly implicate the role of over 170 genes in the colonization of lettuce by this pathogen. I was particularly impressed with the thoroughness with which the authors have addressed the bottlenecks associated with transposon sequencing studies. They are very aware of the limitations of the conclusions that would otherwise result had an insufficient number of transposon insertional mutants been introduced into the plants to start the experiments. I found that the first part of the results did a great job of documenting the substantial number of founder mutants in their mutant library. While the manuscript was generally understandable, there are literally hundreds of places where clunky English grammar, inappropriate words, and other issues detract from understanding the manuscript. As such, the manuscript would definitely benefit from extensive editorial revisions to correct these deficiencies in grammar. Since there have now been several similar studies of the use of transposon sequencing, and more recently Tn barseq, to address the contribution of various genes to the colonization of plants, I was disappointed that the authors did not do a better job of trying to summarize the similarity and differences of their results compared to that of other studies of Pseudomonas, and Ralstonia and other taxa that have been interrogated. I kept thinking that some sort of summary table that would have noted the various genes found by the different studies and the extent to which these genes were found in more than one study would have been an excellent way to have shown both the novelty of any genes found here, but especially to better emphasize the common traits that seem to be involved in colonization of different plant species by different plant pathogens. The extensive discussion of the results seem to over and over show how the same or similar genes had been previously shown to be involved in plant host colonization. This seemed quite repetitive after many such examples, and gave me the impression that the study was largely confirmatory of other work. It seemed like there might have been a better way to have summarized the results as discussed above. It also struck me that it might be most effective to have separately and initially discussed those genes that were found to be contributing to fitness that were unique to this study, before moving on to discuss the many genes that had also been found in other bacteria/plant systems to have been involved in fitness. This would have allowed the novelty of anything found in the study to have been emphasized. As it is now, it is very difficult to know to what extent did any genes found here simply confirm that seen in another systems.
Specifics: Lines 83 through 88. This paragraph seems somewhat disjointed, as the authors talk about leaf surface colonization but then launch into cell wall degrading enzymes and type 2 secretion systems, which would seem to be involved only in the colonization of the apoplast.
Lines 87-88. Again, discussion of biofilms seems out of place at this point.
Line 98. The word should read "benefit" Line 104 "apparition" seems to be some sort of French word that doesn't make sense in this context. It is not an English word that I have ever seen.
Lines 123 through 124. The use of Tnseq addresses only changes in the growth of bacteria, and would not necessarily directly address "virulence" -which smacks of symptoms etc.. Line 136. The authors repeatedly talk about "TA sites". This needs to be better defined.
Lines 149 through 151. The authors are absolutely correct in noting that the number of generations that their mutant library will experience is an essential factor in the success of the study. They should however better explain this to people who wouldn't necessarily understand why this would be the case.
Lines 154 through 156. I do not think it is accurate to suggest that 5 x 10 to the third bacteria had penetrated the leaves by 24 hours. I suspect that many of these cells reflect the multiplication of a smaller number of cells that had initially invaded the leaf. This needs to be clarified, as this might overestimate the size of the founding population.
Lines 157 through 159. The same issue as the above comment. The number of unique cells that entered compared to how many that might have been grown in the interior of the plant needs to be distinguished. Line 162. "Virtually" is the wrong word to be using here.
Line 180. It is very clunky to be referring to 1/10th TSB -this should instead be 10% TSB. Also, in this sentence, and throughout the manuscript, the authors talk about "essential genes" -an inappropriate terminology, as such essentiality is usually defined by the fact that mutants in such genes cannot be recovered because they are absolutely required for growth. What they are really suggesting here is that these genes have a significant effect on fitness, but I would not call them "essential". Lines189 through 200. I have never found that the analysis of GO code frequency is very informative. I did not find this section at all helpful, and would suggest that this paragraph could be eliminated. The GO codes refer to such broad categories of traits that I never found them to be particularly useful.
Lines 218 through 220. More detail should be provided here.
Lines 338 to 339. This introductory sentence seems to jump out of nowhere. And it is unclear how the LPS biosynthesis genes link to anything that were previously discussed.
Line 351. I do not feel it is appropriate to refer to "survival", as they are simply measuring growth, and not survival per se.
Line 359: this is very awkward, because they referred to "growth in the inoculum", whereas they really should be saying "growth of the bacteria in vitro" --since the inoculum refers to the cells that were applied to the plant, rather than being what was grown per se.
Line 372. These studies addressed growth, and not "survival".
Lines 421 through 424. A good example of a sentence which is definitely in need of improved grammar, as it is hard to understand as currently written.
Lines 429-432. Not only does the sentence not make entire sense, but it definitely needs to be expanded upon to be made clearer what the intention of the sentence was.
Line 434. "Screening" is definitely not the word to be used here.
Line 461. More detail needs to be provided in the methods of inoculation as this is critical to understanding the limitations of this experiment.
Lines 467 through 469. It is not clear what they mean by "die-cut". In addition, it would have been quite helpful to have known the proportion of the total recoverable bacterial population that was epiphytic as a function of time after inoculation of the plants. Initially, in the first day or two after inoculation, I would expect that a high proportion of the bacteria would have been on the surface of the leave, whereas later, more would have been apoplastically located. This detail would provide evidence for whether they were actually looking at apoplastic fitness compared to epithetic fitness.
Reviewer #2 (Comments for the Author): In this study, the authors used a previously-generated Xanthomonas hortorum transposon library to examine conditionallyimportant genes for growth in the lettuce apoplast. The resulting lists of conditionally-essential genes in media (TSB) and in planta are broadly useful to comparative studies of plant pathogenic bacteria. Specifically, this manuscript does an excellent job of broadly summarizing the results in the context of functional processes (ie. GO) or metabolic pathways (KEGG) -this is necessary to package these large datasets for the reader. Similarly, as the significance thresholds for these TnSeq analyses are often very stringent, I appreciate the occasional inclusion of genes that presented moderate fitness defects and were likely biologically relevant, even if they did not pass significance cutoffs. Other than some minor comments on the analysis and suggested edits, I do not have major suggestions to improve this manuscript. Table 1. Would expect a skew in read count per insertion site, and therefore median read count might be more informative than mean.
Since the library was spray inoculated, it is curious that motility/chemotaxis genes were not significant in these results. Depending on the inoculation method, these functions may not be needed, but they usually are required to move into the apoplast. Any hypotheses for this result?
226. Comparison to fungal plant pathogen gene expression is possibly useful, but it would be more informative to include in planta gene expression from more-closely related plant pathogens. In planta gene expression profiles are available for P. syringae and related Xanthomonas spp.

High functional redundancy -> what about complementation 'in trans' by neighboring cells?
Minor typos: 113. "techniques"? 121. "it allowed to provide" -awkward 161. "5.6" 231/435. "carbohydrates' metabolism" is used, and might read better simply as "carbohydrate metabolism" NB: Line numbers in our answers refer to the "Revised Manuscript.docx" document. Line numbers in the automatically-generated merged PDF may differ.

Reviewer #1 (Comments for the Author):
This manuscript has presented the results of an interesting transposon sequencing study to address those genes that are involved in colonization of lettuce leaves by a Xanthomonas pathogen. The study seems to have been done quite well, and the results clearly implicate the role of over 170 genes in the colonization of lettuce by this pathogen. I was particularly impressed with the thoroughness with which the authors have addressed the bottlenecks associated with transposon sequencing studies. They are very aware of the limitations of the conclusions that would otherwise result had an insufficient number of transposon insertional mutants been introduced into the plants to start the experiments. I found that the first part of the results did a great job of documenting the substantial number of founder mutants in their mutant library. While the manuscript was generally understandable, there are literally hundreds of places where clunky English grammar, inappropriate words, and other issues detract from understanding the manuscript. As such, the manuscript would definitely benefit from extensive editorial revisions to correct these deficiencies in grammar. Since there have now been several similar studies of the use of transposon sequencing, and more recently Tn barseq, to address the contribution of various genes to the colonization of plants, I was disappointed that the authors did not do a better job of trying to summarize the similarity and differences of their results compared to that of other studies of Pseudomonas, and Ralstonia and other taxa that have been interrogated. I kept thinking that some sort of summary table that would have noted the various genes found by the different studies and the extent to which these genes were found in more than one study would have been an excellent way to have shown both the novelty of any genes found here, but especially to better emphasize the common traits that seem to be involved in colonization of different plant species by different plant pathogens. The extensive discussion of the results seem to over and over show how the same or similar genes had been previously shown to be involved in plant host colonization. This seemed quite repetitive after many such examples, and gave me the impression that the study was largely confirmatory of other work. It seemed like there might have been a better way to have summarized the results as discussed above. It also struck me that it might be most effective to have separately and initially discussed those genes that were found to be contributing to fitness that were unique to this study, before moving on to discuss the many genes that had also been found in other bacteria/plant systems to have been involved in fitness. This would have allowed the novelty of anything found in the study to have been emphasized. As it is now, it is very difficult to know to what extent did any genes found here simply confirm that seen in another systems.

A:
The manuscript has been since corrected by a professional English proofreader (see Acknowledgments section l. 630-631). A comparison of the genes we identified as important for the fitness of X. hortorum pv. vitians in lettuce with those identified in previous in planta Tn-seq or RB-Tnseq studies conducted on Pseudomonas syringae (1), Ralstonia solanacearum (2), Agrobacterium fabrum (3) and Dickeya dadantii (4) was performed (see Methods section l. 609-614). Results have been included in Table S2 and genes of X. hortorum pv. vitians with at least one homolog in any of these other pathogens have been summarized in a new table (Table 4). A section highlighting the common and specific traits unveiled by this comparison has been added to replace the conclusion (l.

435-489).
Specifics: Q: Lines 83 through 88. This paragraph seems somewhat disjointed, as the authors talk about leaf surface colonization but then launch into cell wall degrading enzymes and type 2 secretion systems, which would seem to be involved only in the colonization of the apoplast.

Reviewer #2 (Comments for the Author):
In this study, the authors used a previously-generated Xanthomonas hortorum transposon library to examine conditionally-important genes for growth in the lettuce apoplast. The resulting lists of conditionally-essential genes in media (TSB) and in planta are broadly useful to comparative studies of plant pathogenic bacteria. Specifically, this manuscript does an excellent job of broadly summarizing the results in the context of functional processes (ie. GO) or metabolic pathways (KEGG) -this is necessary to package these large datasets for the reader. Similarly, as the significance thresholds for these TnSeq analyses are often very stringent, I appreciate the occasional inclusion of genes that presented moderate fitness defects and were likely biologically relevant, even if they did not pass significance cutoffs. Other than some minor comments on the analysis and suggested edits, I do not have major suggestions to improve this manuscript. Thank you for submitting your revisions in response to the previous round of review. You have documented these changes well and it appears you have addressed all significant concerns with your revisions and new analyses. Your manuscript has now been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below.
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